Monoclonal antibodies recognizing determinants specific for the promastigote stage of Leishmania mexicana

Two monoclonal antibodies (IX-IF9-D8 and IX-5H9-CI) produced to a membrane enriched fraction of Leishmania mexicana amazonensis promastigotes have been demonstrated to be specific for the promastigote (insect) form and not the amastigote (mammalian host) form of the parasite. The antigens recognized by these monoclonal antibodies are not found on amastigotes isolated from infected animals or on amastigotes isolated from a macrophage cell line J774 infected initially with promastigotes. The antigens are not re-expressed by amastigotes cultured at 34°C; however, amastigotes cultured at 24°C that have begun transformation into promastigotes do express these antigens. The level of expression of these antigens in cultures of amastigotes undergoing transformation into promastigotes, increases with time from 16 to 36 h and appears to correlate with the percentage of promastigotes. Two protein molecules with apparent molecular weights of 40 000 and 92 000 have been identified by radioimmune precipitation as associated with L. mexicana promastigote stage specific determinants.     

The effects of carbon dioxide and oxygen upon the growth and in vitro transformation of Leishmania mexicana mexicana

Growth of Leishmania mexicana promastigotes is highly dependent upon O2 tension. There was a strong positive correlation between the level of O2, growth rate and maximum parasite density. Promastigotes under low oxygen tension decreased in size, protein content and motility, and deaths occurred. Changes in the carbon dioxide concentration (0.1-5.0%) had little effect on promastigote growth. Transformation in vivo of L. mexicana amastigotes to promastigotes also required oxygen, but a low level (0.4%) was sufficient for a high percentage of the amastigotes to transform. At high O2 concentrations, transformation was a little speedier but the number of parasites transforming was little affected. A greater effect was found with CO2. At 5%, transformation was much more rapid than at 0.1% and also an even greater percentage of amastigotes transformed within 48 h. The results give some indication that amastigotes are adpated for growth at low oxygen tensions encountered in vivo and that high carbon dioxide levels may act as a trigger for transformation of the amastigote to promastigote after it is taken up by the sandfly.     

Role of macrophage complement and lectin-like receptors in binding Leishmania parasites to host macrophages

This paper reviews briefly work carried out in our laboratory on the relative roles of the macrophage plasma membrane receptor (CR3) for the cleaved third complement component (iC3b) and the mannosyl/fucosyl receptor (MFR) in binding, ingestion and respiratory burst (RB) response elicited by promastigotes versus amastigotes of Leishmania donovani. In the absence of serum soluble inhibitors (mannan, ribonuclease B) of the MFR cause a dose-dependent reduction in the numbers of promastigotes binding to murine resident peritoneal macrophages and in the proportion of bound parasites eliciting a RB response. For amastigotes no consistent reduction in binding in the presence of mannan is observed but the proportion of parasites eliciting a RB is reduced. Serum-independent binding and ingestion of promastigotes, which are good activators of the alternative complement pathway, is also inhibited by the anti-CR3 monoclonal antibody M1/70, by Fab anti-C3, and by an inhibitor of C3 fixation, sodium salicyl hydroxamate. For amastigotes, which are poor activators of the alternative pathway, a lesser effect is observed with all three inhibitors of CR3-mediated binding. The results obtained with these three independent inhibitors provide strong evidence that cleaved macrophage-derived C3 (iC3b), which can be visualised on the parasite surface in electron microscope sections following addition of anti-C3 antibody and a protein A-gold conjugate, mediates binding to CR3. Modulation experiments in which either CR3 or MFR are rendered inaccessible demonstrate that both receptors must be present on the segment of the macrophage membrane with which the parasite makes contact to mediate binding and ingestion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Leishmania amazonensis Amastigotes Trigger Neutrophil Activation but Resist Neutrophil Microbicidal Mechanisms

Neutrophils are the first cells to infiltrate to the site of Leishmania promastigote infection, and these cells help to reduce parasite burden shortly after infection is initiated. Several clinical reports indicate that neutrophil recruitment is sustained over the course of leishmaniasis, and amastigote-laden neutrophils have been isolated from chronically infected patients and experimentally infected animals. The goal of this study was to compare how thioglycolate-elicited murine neutrophils respond to L. amazonensis metacyclic promastigotes and amastigotes derived from axenic cultures or from the lesions of infected mice. Neutrophils efficiently internalized both amastigote and promastigote forms of the parasite, and phagocytosis was enhanced in lipopolysaccharide (LPS)-activated neutrophils or when parasites were opsonized in serum from infected mice. Parasite uptake resulted in neutrophil activation, oxidative burst, and accelerated neutrophil death. While promastigotes triggered the release of tumor necrosis factor alpha (TNF-α), uptake of amastigotes preferentially resulted in the secretion of interleukin-10 (IL-10) from neutrophils. Finally, the majority of promastigotes were killed by neutrophils, while axenic culture- and lesion-derived amastigotes were highly resistant to neutrophil microbicidal mechanisms. This study indicates that neutrophils exhibit distinct responses to promastigote and amastigote infection. Our findings have important implications for determining the impact of sustained neutrophil recruitment and amastigote-neutrophil interactions during the late phase of cutaneous leishmaniasis.     

Activity of the julocrotine, a glutarimide alkaloid from Croton pullei var. glabrior, on Leishmania (L.) amazonensis

The antiproliferative effect of julocrotine, an alkaloid isolated from Croton pullei var. glabrior (Euphorbiaceae), was studied in the macrophage amastigote and promastigote stages of the protozoan Leishmania (L.) amazonensis, which causes cutaneous leishmaniasis in the New World. Julocrotine showed a dose-dependent effect against the amastigote and promastigote forms, where 79 μM julocrotine inhibited promastigote growth by 54%, with an IC50 of 67 μM. To analyze the antiamastigote activity of the drug, murine peritoneal macrophages infected with L. amazonensis promastigotes were treated with different concentrations of julocrotine. An 80% inhibition of amastigote development was observed using 79 μM julocrotine for 72 h, with an IC50 of 19.8 μM. In addition, ultrastructural observation of the parasites showed a significant reduction in the number of amastigotes in the parasitophorous vacuoles and morphological changes in promastigotes, such as swelling of the mitochondrion, chromatin condensation, presence of membranous structures near the Golgi complex, and some vesicle bodies in the flagellar pocket. A colorimetric assay (MTT), which measures cytotoxic metabolic activity, showed that macrophages maintain their viability after treatment with the drug. These results suggest that julocrotine effectively inhibits the growth of parasites and does not have any cytototoxic effects on the host cell.     

Effect of protein kinase inhibitors on the growth, morphology, and infectivity of Leishmania promastigotes

The effect of two protein kinase inhibitors, staurosporine and H-7, on the growth, morphology and infectivity of Leishmania major and Leishmania amazonensis promastigotes was examined. Incubation with H-7 (600 μM) for up to one hour had no effect on parasite growth, morphology or infectivity. Staurosporine, however, was cytotoxic for promastigotes and incubation for 1, 5 or 15 minutes with 10 μM inhibitor killed 19, 34 and 59 %, respectively, of the parasites. Longer incubations, up to one hour, at this concentration did not increase parasite killing. However, treatment with 25 μM staurosporine for one hour was highly toxic, only 4 % of the promastigotes surviving after 72 h. Lower concentrations of staurosporine, 0.25 and 2.5 μM, had only minor effects on parasite growth. Incubation of either L. major or L. amazonensis with staurosporine (10 μM for 10 minutes) caused marked morphological changes in the size and appearance of the flagellar pocket, and/or cytoplasm of the viable parasites. Treated parasites were still capable of infecting mouse peritoneal macrophages and causing disease in BALB/c mice, though the treated parasites were less virulent than control promastigotes. These results indicate that staurosporine, while inhibiting promastigote growth, does not prevent differentiation to amastigotes and amastigote replication. Received: 26 June 1996 / Accepted: 20 August 1996     

Cutaneous host defense in leishmaniasis: interaction of isolated dermal macrophages and epidermal Langerhans cells with the insect-stage promastigote.

Leishmania species are obligate intracellular pathogens of mononuclear phagocytes. Successful infection depends on sequestration of the promastigote (insect form) within host cells, allowing transformation into the relatively hardy amastigote stage. Promastigotes are killed readily by circulating phagocytes and nonimmune serum, suggesting that cutaneous infection is initiated within a permissive cell in the epidermis or dermis. From large sections of primate skin dermal macrophages and epidermal Langerhans cells were isolated, and their interaction with promastigotes of Leishmania major was investigated in vitro. Dermal macrophages were readily infected with promastigotes, and successful transformation to and replication of amastigotes was observed. Ingestion of promastigotes by dermal macrophages was not associated with a significant respiratory burst, in contrast to that by other macrophage populations, and was associated with significantly greater survival of parasites. Stimulation of these cells with phorbol myristate acetate or opsonized zymosan revealed that those cells were generally oxidatively deficient. Langerhans cells could not be successfully infected by promastigotes under similar conditions. Examination of these cells for expression of CR3, which has been identified as a potential Leishmania receptor, revealed that Langerhans cells did not express the alpha M subunit of CR3, whereas dermal macrophages were CR3 positive. These data support the concept that dermal macrophages are the site of initiation of Leishmania infection.     

In vitro parasite-monocyte interactions in human leishmaniasis: possible role of fibronectin in parasite attachment.

Leishmania spp. must attach to mononuclear phagocyte surfaces before entering this host cell. We investigated the potential role of fibronectin in facilitating parasite attachment. Human plasma fibronectin bound to axenically cultured promastigotes, and promastigotes and amastigotes preferentially bound to fibronectin-coated cover slips. Promastigotes grown in the absence of fibronectin were strikingly deficient in their ability to attach to human monocytes compared with promastigotes grown in the presence of fibronectin. Rabbit anti-human plasma fibronectin antiserum decreased promastigote and amastigote attachment to monocytes. Immunoglobulin G F(ab')2 and Fab fragments also reduced the ability of amastigotes to bind to monocytes. Antiserum pretreatment of amastigotes followed by washing resulted in reduced parasite binding, whereas antibody pretreatment of monocytes did not. Addition of exogenous fibronectin did not enhance parasite attachment to monocytes. These findings suggest that Leishmania spp. can bind fibronectin and may utilize this glycoprotein to facilitate attachment to the mononuclear phagocytes that they infect.     

Freeze-fracture and cytochemistry study of the interaction between Leishmania mexicana amazonensis and macrophages

The process of interaction between macrophages and promastigote and amastigote forms of Leishmania mexicana amazonensis was analyzed using freeze fracture and cytochemistry. The promastigotes inside endocytic vacuoles of macrophages presented an altered distribution of intramembranous particles and a wavy aspect of the plasma membrane. However, amastigotes did not show such alterations. The membrane alterations are probably caused by intracellular cell lysis of the promastigotes by the macrophages. An accumulation of intramembranous particles was seen in the plasma membrane of amastigote forms in the area of adhesion to the macrophages. The parasitophorous vacuole membrane had intramembranous particles randomly distributed. The enzyme activity of Mg++-ATPase, 5'-nucleotidase and NAD(P)H-oxidase was cytochemically detected, at the ultrastructural level, in normal mouse peritoneal macrophages and in macrophages infected with Leishmania mexicana amazonensis. Mg++-ATPase and 5'-nucleotidase are uniformly distributed throughout the macrophage's plasma membrane but were not detected in the membrane lining endocytic vacuoles containing ingested parasites (parasitophorous vacuole). NAD(P)H-oxidase activity was seen in those portions of the macrophage's plasma membrane which enter in direct contact with parasites and also in association with the membrane of the parasitophorous vacuole. The amount of reaction product, indicative of NAD(P)H-oxidase activity, was larger in macrophages which interacted with the promastigote than in those which interacted with the amastigote form of L. mexicana amazonensis. Concanavalin A binding sites and anionic sites of the macrophage's surface, labeled before the interaction, are not interiorized together with the parasites, however, are observed in endocytic vacuoles which do not contain parasites.     

Early response gene expression during differentiation of cultured Leishmania donovani

The promastigote form of the unicellular parasite, Leishmania donovani, must differentiate into the amastigote form to establish an infection in a mammalian host. Identification of genes whose expression changes during differentiation could help reveal mechanisms of Leishmania gene regulation and identify targets for controlling the diseases caused by this human pathogen. Two genomic clones were isolated, P9 that is more highly expressed in promastigotes than in axenic amastigotes and A14 that is preferentially expressed in axenic amastigotes. Analysis of the DNA sequences revealed open reading frames that would encode 55.5 kDa and 100 kDa proteins, respectively, with no homology to known proteins. The mRNA level for these genes during 24 h time courses of parasite differentiation in culture was compared to two genes known to be differentially expressed, c-lpk2 and mkk. Changes in RNA level occurred within 2 h for each gene and continued in advance of morphological changes. The expression levels of these four genes in axenic amastigotes correlated with results from animal-derived parasites.     

Increased infectivity of stationary-phase promastigotes of Leishmania donovani: correlation with enhanced C3 binding capacity and CR3-mediated attachment to host macrophages.

This study demonstrated that the greater infectivity of stationary-phase promastigotes of Leishmania donovani is related to increased complement fixation on the parasite surface, resulting in increased binding to host mononuclear phagocytes (MPs) via complement type 3 receptors (CR3). The in vivo infectivity of log- and stationary-phase promastigotes was compared by measuring parasite loads in the livers of BALB/c mice 14 days after i.v. inoculation. The same populations were tested for their ability to bind to resident murine peritoneal macrophages (RPM) in vitro during a 20-min serum-free incubation period. Stationary-phase parasites displayed both higher in vivo infectivity and increased in vitro binding. However, following uptake by RPM, no significant difference in the 72 hr survival of the two populations could be detected. The in vitro binding of log and stationary parasites was uniformly inhibited in the presence of a mAb (M1/70) specific for CR3, confirming that the interaction of this receptor with its ligand, iC3b, plays a vital role in initial attachment of both promastigote populations. Following incubation with a human serum source, the amount of ligand appeared to be greater on the surface of stationary-phase promastigotes, as indicated by their ability to trigger the alternative complement pathway and by solid-phase ELISA measurements using antiserum specific for human C3. Collectively, these findings suggest that the infectivity of L. donovani promastigotes is influenced by the extent of initial attachment to host MPs, as determined by the levels of complement deposition and subsequent CR3-mediated binding.     

Monoclonal antibodies that recognize distinct epitopes of the macrophage type three complement receptor differ in their ability to inhibit binding of Leishmania promastigotes harvested at different phases of their growth cycle.

The macrophage receptor CR3 has been shown by several investigators to be involved in the binding of Leishmania promastigotes to host macrophages. This receptor is known to recognize iC3b and to mediate direct lectin-like attachment of particles such as yeast zymosan. In the present study, two anti-CR3 monoclonal antibodies, M1/70 and 5C6, which ligate different epitopes of murine CR3, have been used in conjunction with sodium salicyl hydroxamate (Saha; inhibits covalent ester linkages of C3 to an activator surface) to block binding of L. donovani and L. major promastigotes harvested at different phases of their growth cycle. M1/70 inhibited all promastigote binding. 5C6 and Saha blocked in parallel only the binding of peanut agglutinin (PNA)-positive late log and early stationary phase parasites. These results suggest that the binding PNA-positive parasites to CR3 is iC3b-mediated, while entry of the more infective PNA-negative late stationary phase promastigotes into host macrophages may involve direct lectin-like binding to CR3.     

Fast high yield of pure Leishmania (Leishmania) infantum axenic amastigotes and their infectivity to mouse macrophages

Leishmania (L.) infantum (syn. Leishmania chagasi) is a dimorphic protozoan parasite that lives in promastigote and amastigote form in its sandfly vector and mammalian hosts, respectively. Here, we describe an in vitro culture system for the generation of a pure population of L. infantum axenic amastigotes after only 4 days incubation in culture medium supplemented with fetal calf serum, human urine, l-glutamine, and HEPES at 37oC (pH 5.5). Ultrastrutural analysis and infection assays in two macrophage populations (Kupffer cells (KUP) and peritoneal macrophages (PM)) infected with axenic amastigotes demonstrated that they maintained morphological and biochemical (A2 expression) features and a similar infection pattern to tissue-derived L. infantum amastigotes. The susceptibility of the macrophage lines to axenic or tissue-derived amastigotes and promastigotes was investigated. We found a completely different susceptibility profile for KUP and PM. Liver macrophages, both KUP and immigrant macrophages, are intimately involved in the response to L. infantum infection; this difference in susceptibility is probably related to their capacity to eliminate these parasites. Our in vitro system was thus able to generate axenic amastigotes that resemble tissue-derived amastigotes both in morphology and infectivity pattern; this will help in further investigation of the biological characteristics of the host–parasite relationship as well as the process of pathogenesis.     

Role of beta-D-galactofuranose in Leishmania major macrophage invasion

The role of glycosylinositol phospholipid 1 (GIPL-1) of Leishmania (Leishmania) major in the interaction of promastigotes and amastigotes with macrophages was analyzed. Monoclonal antibody MEST-1, which recognizes glycolipids containing terminal galactofuranose (Galf) residues (E. Suzuki, M. S. Toledo, H. K. Takahashi, and A. H. Straus, Glycobiology 7:463-468, 1997), was used to detect GIPL-1 in Leishmania by indirect immunofluorescence and to analyze its role in macrophage infectivity. L. major promastigotes showed intense fluorescence with MEST-1, and GIPL-1 was detected in both amastigote and promastigote forms by high-performance thin-layer chromatography immunostaining by using MEST-1. Delipidation of L. major promastigotes with isopropanol-hexane-water eliminated the MEST-1 reactivity, confirming that only GIPL-1 is recognized in either amastigotes or promastigotes of this species. The biological role of GIPL-1 in the ability of L. major to invade macrophages was studied by using either Fab fragments of MEST-1 or methylglycosides. Preincubation of parasites with Fab fragments reduced macrophage infectivity in about 80% of the promastigotes and 30% of the amastigotes. Preincubation of peritoneal macrophages with p-nitrophenyl-beta-galactofuranoside (10 mM) led to significant ( approximately 80%) inhibition of promastigote infectivity. These data suggest that a putative new receptor recognizing beta-D-Galf is associated with L. major macrophage infectivity and that GIPL-1 containing a terminal Galf residue is involved in the L. major-macrophage interaction.     

免疫小鼠及正常小鼠巨噬细胞对利什曼无鞭毛期的吞噬作用

内脏利什曼原虫主要寄生在巨噬细胞系统的单核吞噬细胞内,在一般情况下其无鞭毛期能抵抗巨噬细胞的杀灭作用。 为了观察经杜氏利什曼原虫免疫后的小鼠其巨噬细胞的作用,我们采用了CFW纯系小鼠,经不同免疫方法于免疫后不同时间观察了体外培养中巨噬细胞的吞噬功能。实验采用的巨噬细胞与杜氏利什曼原虫前鞭毛期的比例为1:4。从每24小时吞噬功能的结果表明,经利什曼鞭毛体纯抗原免疫及福氏佐剂加利什曼抗原免疫的两组小鼠,均以免疫后3周的吞噬率最高,分别为72％及96％;两组吞噬指数的均值±SD(4.46±1.72,6.99±4.36)亦较正常组小鼠(1.68±1.25,1.72±1.15)为高,并具有显著差异(P<0.05)。提示了特异性抗原以及与佐剂合并具有对吞噬功能的激活作用。实验并观察了巨噬细胞内利什曼原虫无鞭毛期的活力作用,从吞噬原虫后20小时开始至 144小时,正常小鼠巨噬细胞内的无鞭毛期再经三恩氏培养基培养后均能恢复为前鞭毛期,而经免疫小鼠巨噬细胞内的利什曼原虫无鞭毛期在72小时后即消失活力。 另外,对小鼠腹腔巨噬细胞吞噬利什曼原虫的动态亦作了仔细观察。 实验结果说明了经过免疫的小鼠,由于被淋巴细胞激活后的巨噬细胞能杀死利什曼原虫,巨噬细胞在宿主对感染应答中是一个重要部分,对于探索黑热病的免疫机理具有一     

Leishmania braziliensis promastigotes and amastigotes interact differently with host macrophages

In vitro and in vivo ultrastructral studies reveal that the parasite entrance into the macrophage occurs by phagocytosis. The early stage of phagocytosis exhibited different ultrastructural characteristics in both forms of the parasite. Long and prominent projections from peritoneal exudate macrophages made focal contacts with the promastigote surface. The amastigotes, in turn, laid on cup-shaped extensions of the macrophage membrane. Later stages of the phagocytosis are characterized by progressive and complete engulfment of both promastigotes and amastigotes.     

Changes in the proteome and infectivity of Leishmania infantum induced by in vitro exposure to a nitric oxide donor

Leishmania species are protozoan parasites that exhibit an intracellular amastigote form within mammalian macrophages and an extracellular promastigote form inside the sandfly vector. The generation of nitric oxide (NO) upon activation of macrophages is surely the principal killing effector of intracellular amastigotes but little is known about the potential action of NO against the promastigote phase during its multiplication inside the digestive tract of the sandfly vector. Therefore, we have approached this issue by using an in vitro model to study the effect of an NO donor, 3-morpholinosydnonimine (SIN-1), on the proteome and infectivity of promastigotes of Leishmania infantum. Exposure of promastigotes to SIN-1 during its logarithmic growth phase caused a dramatic effect on parasite protein expression and viability, consequently killing about 60-70% of the promastigotes. The significant changes in the proteome included the over-expression of enolase, peroxidoxin precursors, and heat-shock protein 70 (HSP70), under-expression of 20S proteasome alpha 5 unit, and phosphomannomutase and induced expression of 3-hydroxy-3-methyglutaryl-CoA (HMG-CoA) synthase and prostaglandine f2-alpha (PGD2) synthase. Interestingly, promastigotes that resisted treatment showed enhanced infectivity to J774 macrophages in comparison to the controls. This finding together with the appearance of the PGD2S and an over-expression of HSP70 isoforms in treated promastigotes led us to speculate the existence of NO-mediated programmed cell death (PCD) events as a potential mechanism of population regulation and selection of properly infecting forms that predominantly operate on the promastigote stage.     

Large-scale production of amastigotes by a human monoblastoid cell line

In this study, a human monoblastoid cell line (TPH-1) was tested in vitro for the production of Leishmania amastigotes. The number of TPH1 cells increased with time and 6 days after promastigote infection the percentage of infected cells was around 45%. Pre-treatment of TPH1 cells with retinoic acid induced the cells to differentiate into unreplicating macrophage-like cells. Ninety per cent was parasitized 6 days after promastigote infection; the number of amastigotes quintuplied during this period of time; this result was irrespective of the Leishmania species used for experiments. Viable and infective parasites were obtained from treated and nontreated cells. TPH1 cells merit further consideration for research concerning new molecules active against Leishmania.